In order that a protein as a main part responsible to the life function of a living body exerts its function orderly in the cell population, modification after translation including modification of a glycan has an extremely important role. In recent years, it has been found continuously that almost of proteins in living bodies undergo modification by glycans and that glycans added to proteins play an important role in various fields of life phenomena such as infection of virus, parasitism and infection of protozoa, binding of toxin, binding of hormone, conception, emergence and differentiation, stability of protein, cancer cell metastatsis, and apoptosis.
For analyzing a glycan function, the structural analysis for the glycan is first indispensable. It is expected that more importance will be attached to the glycan structure analysis method in the future. However, since the structural analysis of the glycan requires enormous time, energy, and experience it has been expected develop a system capable of extracting the features of various glycan structures more conveniently, quickly, at a higher sensitivity and higher accuracy and capable of distinguishing them from each other, instead of aiming at a complete determination of structure based on the existent method.
A micro-array is a collective name of instruments in which immobilized specimens such as various types of DNA-proteins are immobilized spot-wise at a high density on a solid phase carrier (glass membrane silicon chip) and this can detect the presence or absence of molecules binding specifically to various types of immobilized specimen spots (hereinafter referred to as a probe). For the probe molecules, those labeled with fluorescence are generally used and, by reacting a probe solution with an array surface and then observing by a fluorescent detection scanner, quantitative analysis can be conducted for the probe molecules binding to each of specimen spots. Since a DNA micro-array was developed by Affymetrix Co in USA, micro-arrays have been used in an extremely wide variety of fields of study to provide various novel findings to human beings.
For the study of structural and functional information on glycans which are referred to as the third life chain, in a case where interaction of glycans and proteins showing interaction with the glycan (glycan binding protein, for example, lectin) can be analyzed quickly at a high sensitivity in a large scale by utilizing the micro-arrays, it is considered that this may provide an extremely useful tool that can be utilized in a wide range from the basic study to medical diagnosis or industrial application.
It has been known that binding between the glycan and the protein that exhibits the interaction with the glycan is generally a weak interaction compared with a general dissociation constant of an antigen-antibody reaction (Kd=10−8 M or less), and the dissociation constant (Kd) thereof is often 10−6 M or more. Further, it has been known that binding between the glycan and the protein that exhibits the interaction with the glycan comprises relatively fast dissociation and association reaction and, as a result, the equilibrium tends to direct to the dissociation side by a cleaning operation or the like compared with general inter-protein interaction or interaction between complementary nucleotide fragments. For example, also in a case of purifying a lectin by a glycoside protein immobilization column or the like, when the lectin binding is weak, a phenomenon has often been observed that lectin runs off to the outside of a column during the cleaning operation.
In the general existent micro-array technique using a slide glass, after the process of bringing a probe solution into contact with an immobilizing specimen to cause binding reaction, operations of cleaning and removing the probe solution and completely removing the water content deposited on the slide glass are conducted by using a jet gas or a centrifugator, and then imaging is conducted by using a micro-array scanner. This is because fluorescence on the slide glass can not be observed by a general micro-array reader in a state where the water content is deposited. Even when the probe solution is removed in a stage before the scanning, it is considered that the dissociating reaction of the probe molecules does not proceed easily since the dissociation rate constant is sufficiently small in the interaction showing strong bond such as complementary nucleotide fragment or antigen-antibody reaction. However, upon observation of the interaction of high, dissociation rate constant, that is, weak interaction shown generally between the glycan and the protein that exhibits interaction with the glycan, the dissociation reaction proceeds between the glycan and the protein that exhibits interaction with the glycan at the instance of conducting the removing and cleaning operation for the probe solution and it is difficult to obtain an accurate interaction information under the equilibrium state. Accordingly, in the micro-array, the cleaning operation of the probe solution results in a significant problem in a case of precisely analyzing the interaction information in the equilibrium state between the glycan and the protein that exhibits interaction with the glycan.
DNA micro-arrays have been utilized in an extremely wide range at present. Also for protein micro-arrays, future utilization is expected in the field of basic study such as clarification of the function of a protein as the product of DNA transcription in the living body, as well as in the application field such as diagnosis or judgment on the basis of quantitative or qualitative change of the protein, and vigorous researches have been conducted all over the world in view of the study. However, development and popularization for the protein micro-array is greatly delayed compared with that for the DNA micro-array. As one of the causes, it has been pointed out by various workers that a step of immobilizing protein specimens having various different natures at a predetermined ratio in a state of keeping the activity as it is extremely difficult technically.
For the methods of immobilizing proteins on arrays, a method developed in the earliest stage includes a method of physically adsorbing a protein on a membrane typically represented by a PVDF (Non-Patent Document 1). It has been reported that some proteins such as transcription factors can maintain the activity to some extent but it lacks in generality. Further, in a case of immobilization on the membrane, increase in the density of the array has been limited. For obtaining higher density, while a study has been progressed in the direction of immobilizing the protein to the surface of a solid such as metal or glass, the protein generally has a nature tending to be denatured by the contact with the surface of the solid such as metal or glass. Accordingly, an immobilizing method using a certain linker for crosslinking the surface of the solid and the protein has been studied and developed vigorously.
An example of the method of mitigating the problem of denaturation of the protein includes a method of joining a pad of a polyacrylamide of 10 to 100 μm thickness on a slide glass and spotting the protein thereto (Non-Patent Documents 2 and 3). In this case, since the protein is immobilized in a three-dimensional space, it is said that improvement by 100 times or more may be expected in view of the quantity compared with a method of immobilizing on a two-dimensional surface. Further, there is also a method of immobilizing the protein by way of an amino group in a porous polyacrylamide gel (Non-Patent Document 4). However, the methods described above have not yet been popularized generally since it is necessary to prepare a expensive and special slide glass. Further, the immobilized protein layer has a thickness and this is sometimes not preferred depending on the detection method.
Further, Patent Document 4 discloses a photoconductive substrate having a closed type reaction vessel with spots of glycan binding protein, and teaches technology to introduce light from one side edge of the substrate so as to detect fluorescence excited by evanescent waves generating on the surface of substrate.
However, reagent is supplied into the vessel only by using capillary action, since the reaction vessel is formed in a closed gap between two layered glass plates. That is, it wastes long time, reagent can not be supplied to the vessel momentarily, especially under monitoring condition.
Since the light is introduced from one side edge of the substrate, intensity of scattered light of evanescent wave at the one side of substrate is higher than intensity at another side.
Therefore evanescent waves generated on the surface of the substrate can not be uniform, and measurement accuracy is reduced.
Methods of immobilizing proteins to the solid phase that have been being studied most vigorously at present include a method of expressing a protein in a form attached with some or other tag and immobilizing the protein by way of its tag portion to a solid phase carrier. It has been said for the method that it can provide an effect of improving the effective ligand concentration of the protein or alignment for the direction of the protein in principle. An example of such method includes a method of immobilization by way of oligohistidine tag to a substrate modified at the surface with a nickel complex (Non-Patent Document 5) or a method of immobilization by way of avidin-biotin (Patent Document 1).
It is considered that such methods are effective in immobilization while keeping the activity of the protein as it is, or making the rate of immobilization uniform. However, since it is considered to require much cost and enormous labor for adding tags at the gene level to all proteins to be immobilized on the micro-array and conducting expression by Escherichia coli bacteria or non-cell systems and purification, it is difficult for usual workers to easily utilize the method optionally and in the form conforming with individual requirements.
On the contrary, a method of utilizing functional groups of proteins for immobilization with solid phase carrier has a feature that proteins as extracted from natural products or commercial protein specimens can be immobilized as they are and utilized for the micro-array. A method of immobilization by way of amino groups in proteins to a solid phase carrier includes a method of immobilizing a protein by way of active ester groups bonded on the surface of the solid phase, or a method of immobilizing a protein by way of epoxy groups arranged on the surface of the solid phase (Non-Patent Document 6). While the method of immobilization by way of the amino groups of the protein is a simple method, since commercial proteins, bio-body extract ingredients, or recombinant proteins with no particular tags can also be immobilized simply and conveniently, individual users can optionally select proteins in accordance with the object of their own and can use them with optimization to micro-arrays conforming with the purpose rapidly and at a low cost. Drawbacks of the immobilizing method by way of the amino groups of the protein include, for example, that the number of lysine residues in the proteins is different depending on individual proteins or that inactivation of the protein may possibly occur depending on the position of the lysine residues used for immobilization.    Patent Document 1: JP-A No. 2001-520104    Patent Document 2: JP-A No. 8-201382    Patent Document 3: JP-W No. 2002-544485    Non-Patent Document 1: L. J. Holt, K. Bussow, G. Walter, I. M. Tomlinson, Nucleic Acids Res. 15, E72, 2000    Non-Patent Document 2: D. Guschin, G. Yershov, A. Zaslavsky, A. Germmell, V. Shick, D. Proudnikov, P. Arenkov, A. Mirzabekov, Anal. Biochem., 250, 203-211, 1997    Non-Patent Document 3: A. Lueking, M. Horn, H. Eickhoff, K. Bussow, H. Lehrach, G. Walter, Anal. Biochem., 270, 103-111, 1999.    Non-Patent Document 4: P. Mitchell, Nat. Biotechnol., 20, 225-229, 2002    Non-Patent Document 5: H. Zhu, M. Bilgin, R. Bangham, D. Hall, A. Casamayor, P. Bertone, N. Lan, R. Jansen, S. Bidlingmaier, T. Houfek, T. Mitchell, P. Miller, R. A. Dean, M. Gerstein, M. Snyder, Science, 293, 2101-2105, 2001    Non-Patent Document 6: H. Zhu, J. F. Klemic, S. Chang, P. Vertone, A. Casamayor, K. G. Klemic, D. Smith, M. Gerstein, M. A. Reed, M. Shyder, Nat. Genetics. 26, 283-289, 2000